ABSTRACT
We have demonstrated that a trypsin sensitive enzyme such as L-asparaginase can be rendered trypsin resistant by genetically fusing its gene with that of a single-chain antibody derived from a preselected monoclonal antibody capable of providing protection against trypsin. The chimeric L-asparaginase retained 75% of its original activity upon exposure to trypsin, whereas the native unprotected L-asparaginase control was totally inactivated.
Subject(s)
Asparaginase/genetics , Protein Engineering , Trypsin/pharmacology , Antibodies, Monoclonal/genetics , Asparaginase/drug effects , Base Sequence , Molecular Sequence Data , Recombinant Fusion Proteins/drug effects , Trypsin/immunologyABSTRACT
Evidence for a small size protein (ca. 3500 kDa) exhibiting epitopic homology to the Atlantic winter flounder antifreeze protein (AFP) is found in the snow molds Coprinus psychromorbidus, Myriosclerotinia borealis, and Typhula incarnata. The protein shows strong cross-reactivity with antisera specific for the flounder AFP. Preliminary studies suggest that the protein is synthesized in response to lowering the culture temperature, and that it is membrane associated and, therefore, may function in an analogous capacity to the fish AFP. Also, the protein is shown to have antifreeze properties as determined by nuclear magnetic resonance microimaging experiments.
Subject(s)
Agaricales/chemistry , Coprinus/chemistry , Fungal Proteins/chemistry , Glycoproteins/chemistry , Sequence Homology , Animals , Antifreeze Proteins , Blotting, Western , Flounder , Freezing , Isoelectric Point , Molecular WeightABSTRACT
A monoclonal antibody against wheat germ sucrose synthetase is developed and characterized. Its use in studying the effect of cold acclimation on the expression of sucrose synthetase in winter and spring wheat plants is described. The antibody shows cross-reactivity with sucrose synthetase from maize and pea plants, as well as carrot cells. A gradual accumulation of the enzyme as a function of time spent at 2 degrees C is observed in both wheat varieties. In contrast, an initial sharp rise in the mRNA level is observed, which agrees with the previously reported response of maize plants subjected to anaerobic stress.